Control of gene expression in eukaryotes

ABSTRACT

A chemically inducible gene expression system is described. A chimeric gene having a first sequence comprising a promoter and a regulator polypeptide is linked with a second sequence comprising a promoter and a coding or non-coding sequence. Expression of the target gene of the second sequence is controlled by the regulator polypeptide which is acted upon by an inducer. The inducer is a chemical compound, such as OHP, which acts upon the OHP responsive regulator polypeptide, which can be obtained from  Rhodococcus  sp. V49. Various domain regions and complementary response elements are also described.

1. INTRODUCTION

This invention relates to an inducible gene expression system,particularly but not exclusively eukaryotes, such as plants, forexample.

2. BACKGROUND TO THE INVENTION

Manipulation of plants to improve certain characteristics requires thecontrol of expression of foreign or endogenous genes in plant tissues.Such manipulation relies on the availability of mechanisms to controlgene expression as required. It is therefore advantageous to have thechoice of a variety of different promoters so that the most suitablepromoter may be used. A range of promoters is known to be operativewithin plants.

Within the promoter there are several defined domains which arenecessary for the function of the promoter. The first of these domainsis located immediately upstream of the structural gene and forms thecore promoter region, about 70 base pairs immediately upstream of thegenes. This region contains the CAAT and TATA boxes and represents atranscription initiation sequence which defines the transcription startsite for the gene. A series of regulatory sequences upstream of the corepromoter sequence constitute the remainder of the promoter. Theregulatory sequences determine the expression levels, the spatial andtemporal pattern of expression and possible expression under inductiveconditions.

The control of expression of heterologous genes in plant cells isimportant for the successful genetic manipulation of plants to alterand/or improve phenotypic characteristics. Promoters and/or regulatorysequences from bacteria, viruses, fungi and plants have been used tocontrol gene expression in plants. In some cases it will be desirable tocontrol the time and/or extent of the expression of introduced geneticmaterial in plants, plant cells or tissue. The ability to regulate theexpression of transgenes provides several important advantages: (1)regulation of expression of gene(s) that might interfere with thetransformation and regeneration process (Roeder et al., 1994, McKenzieet al., 1998), (2) reversible control of gene expression at a specifictime (e.g. manipulation of carbon metabolism by Caddick et al., 1998 andsecondary product formation by Sommer et al., 1998), (3) control ofgrowth and development (e.g. flowering, plant fertility, cell wallformation), (4) control of genes that respond to environmental signals(e.g. attack by pathogens, such as, for example, nematodes, arachnids oraphids), (5) expression of selectable marker genes and (6) expression ofrecombinase proteins at specific time points. Each of these applicationscan use the inducible gene expression system and novel sequences of thepresent invention.

2.1 Known Regulatable Gene Expression Systems in Plants

A few plant genes are known to be induced by a variety of internal andexternal factors including plant hormones, heat/cold shock, chemicals,pathogens, lack of oxygen and light. Few of these systems have beendescribed in detail.

Ideally a chemically inducible activating promoter in a 5′ regulatoryregion should have low background activity in the absence of an inducerand demonstrate high expression in the presence of an inducer. Achemically inducible repressing promoter in a 5′ regulatory regionshould have low background activity in the presence of an inducer anddemonstrate high expression in the absence of an inducer. Theactivator/repressor should also only allow control of the transgene.This renders the use of most endogenous promoters unsuitable and favorsthe use of those better characterized regulatory elements of modelorganisms distant in evolution, such as yeast, E. coli, Drosophila ormammalian cells, that respond to signals that are usually notencountered in higher plants. These characteristic regulatory elementsare, however, less advantageous in their operation than the systemproposed in the present invention. On this basis, two different conceptsof gene control can be realized, namely promoter-repressing systems andpromoter-activating systems.

2.2 Promoter-Repressing Systems

The repression principle is based on the sterical interference of aprotein with the proteins important for transcription. It is a commonmechanism in bacteria, for example LexA, Lac and Tet, but occurs muchless frequently in higher eukaryotes. Two bacterial repressor/operatorsystems (Lac and Tet) have been used to control the activity ofpromoters transcribed by RNA polymerase II. Gatz and Quail (1988) taughtthe use of the Tn10-encoded Tet repressor/operator with a cauliflowermosaic virus 35S promoter in a transient plant expression system.Frohberg et al., (1991) and Gatz et al., (1991, 1992) characterised theeffect of placing Tet operator sequences at different positions in aCaMV 35S promoter. U.S. Pat. No. 5,723,765 and International PatentApplication, Publication No. WO 96/04393 disclosed use of the Tetrepressor system for the inducible expression of the Cre recombinase intransgenic plants. Wilde et al., (1992) used the Lac repressor/operatorsystem for the inducible expression from a chlorophyll a/b bindingprotein promoter in protoplasts of stably transformed plants.

2.3 Promoter-Activating Systems

A second approach for the construction of a chemically inducible systemis to use transcriptional activators from higher eukaryotes. Themammalian glucocorticoid receptor (GR), which activates eukaryoticexpression only in the presence of steroids has been used by Picard etal., (1988) in Schizosaccharomyces pombe. Schena et al., (1991) haveshown that transcription from a target promoter containing GR-bindingsites was strictly dependent on the addition of steroids in transientlytransformed tobacco cells. Lloyd et al., (1994) have used a fusion ofthe steroid receptor protein with the maize transcription factor R tocomplement an Arabidopsis mutant in a steroid inducible fashion. Aoyamaand Chua (1997) disclosed use of a chimeric transcription factorconsisting of the DNA-binding domain of the yeast transcription factorGal4, the transactivating domain of the herpes viral protein Vp16 andthe receptor domain of the rat glucocorticoid receptor to induce theexpression of a reporter gene in transgenic plants through theapplication of steroids.

International Patent Application, Publication No. WO 96/27673 describesthe use of a steroid receptor system in transgenic plants using chimericGR receptors with Vp16 and C1 transcriptional activation domains andGal4 DNA binding domains.

Another eukaryotic ligand-dependent activator is Ace1, acopper-dependent transcriptional activator from yeast. Mett et al.,(1993) have shown that Ace1 regulates the expression of a suitabletarget promoter (CaMV 35S −90 bp promoter containing the Ace1 bindingsite) in transgenic plants. McKenzie et al., (1998) used a similarsystem (Ace1 binding sites with a CaMV 35S −40 bp promoter) toinvestigate copper-inducible activation of the ipt gene in transgenictobacco.

AlcR is the specific activator of the Aspergillus nidulansethanol-utilisation pathway, mediating the induction of its owntranscription and that of the structural genes alcA and aldA. AlcR is aDNA binding protein that recognises specific binding sites in structuralgene promoters (Kulmburg et al., 1992, Fillinger & Felenbok 1996).Felenbok (1991) used the AlcA-AlcR system for the expression ofrecombinant proteins in Aspergilli. The ethanol inducible gene switchwas used by Caddick et al., (1998) to manipulate carbon metabolism intransgenic plants and also by Salter et al (1998) to examine theinduction of a chloramphenicol acetyltransferase (CAT) reporterconstruct by ethanol. This system has also been used in InternationalPatent Application, Publication No. WO 93/21334 for the inducibleactivation of a chimeric alcA/CaMV 35S promoter in transgenic plants.

2.4 Fusion Proteins

A third strategy is based on the construction of fusion proteins betweentranscriptional transactivation domains and bacterial repressor proteinssuch as the Lac and the Tet repressor. Weinmann et al (1994) used atetracycline controlled transactivator (the virus protein 16 (Vp 16)activation domain fused to the Tet repressor protein) to switch offexpression of a GUS transgene in transgenic plants in the presence ofthe inducer.

2.5 Mutant Repressor Proteins

A fourth strategy is based on the creation of mutant repressor proteinsthat bind to DNA only in the presence of the inducer. Gossen et al.,(1995) have developed a reverse Tet repressor protein that binds to DNAonly in the presence of the inducer and used this system successfully inmammalian cells.

Very recently, in International Patent Application No. PCT/GB98/01893work was carried out at Rhodococcus sp. V49 in respect of biosensormaterials and methods of uses thereof. Rhodococcus sp. V49 (formerlyNocardia corallina) ATCC19070 is a non-acid fast, gram-positiverod-shaped soil bacterium. It can use a range of monoaromatic compounds,including 3-(2-hydroxyphenyl)propionic acid (orthohydroxyphenylpropionicacid, OHP) and 2-hydroxy cinnamic acid as the sole carbon source. It isalso able to grow on n-hexadecane, benzene and toluene. Theinternational patent application, the subject matter of which is to bedeemed incorporated herein, discloses the nucleotide sequence of the 7.5kb OHP operon from Rhodococcus sp. V49.

The polypeptide encoded by the ohpR gene shows a strong sequencesimilarity throughout its length to a number of bacterialtranscriptional regulators from the GntR family (Haydon & Guest 1991).The strong sequence similarity indicates that ohpR encodes a prokaryotictranscriptional regulator.

International Patent Application No. PCT/GB98/01893 discloses the use ofgenetically manipulated mycolic acid bacteria cells solely as sensorsfor analytes in environmental samples. The potential other uses andmodifications of the novel nucleotide sequences described in the presentinvention are nowhere contemplated in PCTI/GB98/01893.

3. SUMMARY OF THE INVENTION

The present invention provides a method of controlling eukaryotic geneexpression comprising transforming a eukaryotic cell with an induciblegene expression system, the gene expression system comprising a firstnucleotide sequence comprising a 5′ regulatory region operably linked toa nucleic acid sequence which encodes a regulator polypeptide and anuntranslated 3′ termination sequence, and a second nucleotide sequencecomprising a 5′ regulatory region operably linked to a nucleic acidsequence which is a coding or non-coding sequence, the expression of thenucleic acid sequence of the second nucleotide sequence being controlledby the regulator polypeptide of the first nucleotide sequence using aninducer, the inducer thereby causing modulation of expression of thenucleic acid sequence of the second nucleotide sequence, and thenucleotide sequence of the regulator polypeptide and/or the 5′regulatory region, parts thereof, of the second nucleotide sequencebeing isolated from a prokaryote source.

The present invention also provides a chimeric gene comprising a firstnucleotide sequence comprising a 5′ regulatory region operably linked toa nucleic acid sequence which encodes a regulator polypeptide and anuntranslated 3′ termination sequence, and a second nucleotide sequencecomprising a 5′ regulatory region operably linked to a nucleic acidsequence which is a coding or non-coding sequence, the expression of thenucleic acid sequence of the second nucleotide sequence being controlledby the regulator polypeptide of the first nucleotide sequence using aninducer, the inducer thereby causing modulation of expression of thenucleic acid sequence of the second nucleotide sequence, and thenucleotide sequence of the regulator polypeptide and/or the 5′regulatory region or parts thereof of the second nucleotide sequencebeing isolated from a prokaryote source.

Advantageously, the regulator polypeptide comprises one or more domains,which domains may be a ligand binding domain, a nucleic acid bindingdomain, a transactivation domain, a targeting domain, asilencing/repressing domain or a dimerization domain. The regulatorsequence may thus comprise a chimeric gene of different sequences.

3.1 Definitions

In order to provide a clear and consistent understanding of thespecification and terms used herein, the following definitions areprovided:

3.1.1 Regulatable Gene

A gene containing at least one regulatable nucleic acid sequence and atleast one associated coding or non-coding nucleic acid sequence. Thegenes may be of natural, synthetic or partially natural/partiallysynthetic origin.

3.1.2 Inducer

An elemental or molecular species which controls, for example,initiates, terminates, increases or reduces, by direct or indirectaction, the activity of a regulatable nucleic acid sequence in a systemin which the inducer is not normally found in an active form in anamount sufficient to effect regulation of transcription, to the degreeand at the time desired, of transcribable nucleic acid sequenceassociated with the regulatable nucleic acid sequence.

This terminology embraces situations in which no or very little induceris present at the time transcription is desired or in which some induceris present but increased or decreased regulation is required to effectmore press transcription as desired. Thus, if the system containing theregulatable nucleic acid sequence is, for example, a transgenic plant,an inducer is a species not naturally found in the plant in an amountsufficient to effect regulation/modulation, and thus transcription of anassociated gene, to the desired degree at the time desired.

By “direct action” it is intended that the inducer action results fromthe direct interaction between the inducer and the nucleic acidsequence. By “indirect action” it is meant that the inducer actionresults from the direct interaction between the inducer and some otherendogenous or exogenous component in the system, the ultimate results ofthat direct interaction being activation or suppression of the activityof the nucleic acid sequence. By “active form” it is intended that theinducer be in a form required to effect control.

3.1.3 Regulator Polypeptide

This term as used herein refers to polypeptides which modulate theexpression of a target gene (the nucleic acid sequence of the secondnucleotide sequence of the present invention) in response to an inducer.The regulator polypeptide may comprise one or more of a ligand bindingdomain, a nucleic acid binding domain, a transactivation domain, atargeting domain, a silencing/repressing domain or a dimerizationdomain.

3.1.4 Chimeric Sequence or Gene

A nucleic acid sequence containing at least two parts, e.g. partsderived from naturally occurring nucleic acid sequences which are notassociated in their naturally occurring states, or containing at leastone part that is of synthetic origin and not found in nature.

3.1.5 Coding Sequence

A nucleic acid sequence which, when transcribed and translated, resultsin the formation of a polypeptide.

3.1.6 Non-Coding Sequence

A nucleic acid sequence which is not transcribed and translated,resulting in the formation of a polypeptide when associated with aparticular coding nucleic acid sequence. Thus, for example, a sequencethat is non-coding when associated with one coding sequence may actuallybe coding when associated with another coding or non-coding sequence.

3.1.7 Plant Tissue

Any tissue of a plant in planta or in culture. This term includes, butis not limited to, whole plants, plant cells, plant organs, plant seeds,protoplasts, callus, cell culture and any groups of plant cellsorganized into structural and/or functional units. The use of this termin conjunction with, or in the absence of, any specific type of planttissue as listed above or otherwise embraced by this definition is notintended to be exclusive of any other type of plant tissue.

3.1.8 Modulation

The increasing or decreasing of the level of expression of a gene or thelevel of transcription of a nucleic acid sequence. The definition is notintended to embrace any particular mechanism.

4. DESCRIPTION OF THE FIGURES

In order that the invention may be easily understood and readily carriedinto effect, reference will now be made, by way of example, to thefollowing diagrammatic drawings, wherein:

FIG. 1 shows a schematic diagram of the plasmid pSK489 as used in thepresent invention. The plasmid contains the nucleotide sequence for ohpR(from nucleotide 295 to nucleotide 1035 of SEQ ID NO: 1) insertedbetween the EcoRI and NotI sites in pBluescript;

FIG. 2 shows a schematic diagram of the plasmid p35SC1 (Tuerck & Fromm1994) as used in the present invention. The plasmid contains the C1 cDNAas described in Paz-Ares et al., (1987) inserted as an EcoRI fragmentbetween a CaMV 35S promoter, Adh1 intron 1 and a CaMV 35S terminator,

FIG. 3 shows a schematic diagram of the plasmid pSK483 as used in thepresent invention. The plasmid contains the C1 coding region asdescribed in Paz-Ares et al., (1987) inserted between the EcoRI and theNotI sites in pBluescript;

FIG. 4 shows a schematic diagram of the plasmid pSK-59 as used in thepresent invention. The plasmid contains part of the ohp operator(nucleotide 1036 to nucleotide 1449 of SEQ ID NO: 1) inserted betweenthe XhoI and SalI sites in pBluescript;

FIG. 5 shows a schematic diagram of the plasmid pSK52040 as used in thepresent invention. The plasmid contains the CaMV 35S promoter, a GUSintron (Vancanneyt et al., 1990) and a CaMV 35S terminator inpBluescript;

FIG. 6 shows a schematic diagram of the plasmid pSK58040 as used in thepresent invention. The plasmid contains the ohp operator from nucleotide1036 to nucleotide 1449 of SEQ ID NO: 1 inserted in plasmid pSK52040into the XhoI site upstream of the CaMV 35S −90 bp core promoter.Downstream of the CaMV 35S core promoter are located a GUS intron and anos terminator;

FIG. 7 shows a schematic diagram of plasmid pDV35 μl as used in thepresent invention. The plasmid contains the CaMV 35S promoter and theCaMV 35S terminator in pBluescript;

FIG. 8 shows a schematic diagram of plasmid pDV60 as used in the presentinvention. The plasmid contains the chimeric promoter of SEQ ID NO: 19,and the CaMV 35S terminator in pBluescript. The chimeric promoter inSeq. ID. 19 contains a 36 bp region of the ohp operon (from nucleotide1225 to nucleotide 1260 of SEQ ID NO: 1) inserted into the CaMV35Spromoter at nucleotide −21;

FIG. 9 shows a schematic diagram of plasmid pSK60040 as used in thepresent invention. The plasmid contains the chimeric promoter describedin FIG. 8 above (Seq. ID. 19), a GUS intron (Vancanneyt et al., 1990)and a nos terminator in pBluescript;

FIG. 10 shows a schematic diagram of plasmid pSK-490 as used in thepresent invention. The plasmid contains the chimeric regulator being atranslational fusion between the ohpR coding sequence (nucleotide 295 tonucleotide 1035 of SEQ ID NO: 1) and part of the C1 cDNA (from the NarIat nucleotide 536 to the end of the coding region at nucleotide 839,amino acids 179 to 279 of the C1 protein) inserted into pBluescriptbetween the HindIII and Not I sites;

FIG. 11 shows a schematic diagram of plasmid pSK491 as used in thepresent invention. The plasmid contains the chimeric regulator being atranslational fusion between the ohpR coding sequence (nucleotide 295 tonucleotide 1035 of SEQ ID NO: 1) and part of the C1 cDNA (from the PstIsite at nucleotide 674 to the end of the coding region at nucleotide839, amino acids 219 to 279 of the C1 protein) inserted into pBluescriptbetween the HindIII and Not I sites,

FIG. 12 shows a schematic diagram of plasmid pUCAP (van Engelen et al.,1995),

FIG. 13 shows a schematic diagram of plasmid pDV35S2 as used in thepresent invention. The plasmid is the pUCAP plasmid with the CaMV 35Spromoter/terminator fragment from pDV35S1;

FIG. 14 shows a schematic diagram of plasmid pSK10489 as used in thepresent invention. The plasmid contains the ohpR sequence inserted intothe BamHI and XbaI sites between the CaMV 35S promoter and the CaMV 35Sterminator in pDV35S1;

FIG. 15 shows a schematic diagram of plasmid pSK10490 as used in thepresent invention. The plasmid contains the translational fusion betweenthe ohpR coding sequence (nucleotide 295 to nucleotide 1035 of SEQ IDNO: 1) and part of the C1 cDNA from the NarI at nucleotide 536 to theend of the coding region at nucleotide 839, amino acids 179 to 279 ofthe C1 protein) from plasmid pSK490 inserted into the BamHI and XbaIsites between the CaMV 35S promoter and the CaMV 35S terminator inpDV35S 1;

FIG. 16 shows a schematic diagram of plasmid pSK10491 as used in thepresent invention. The plasmid contains the translational fusion betweenthe ohpR coding sequence (nucleotide 295 to nucleotide 1035 of SEQ IDNO: 1) and part of the C1 cDNA (from the PstI site at nucleotide 674 tothe end of the coding region at nucleotide 539, amino acids 219 to 279of the C1 protein) from plasmid pSK491 inserted into the BamHI and XbaIsites between the CaMV 35S promoter and the CaMV 35S terminator in pDV35μl;

FIG. 17 shows a schematic diagram of plasmid pBNP as used in the presentinvention. The plasmid is also known as pBINplus (van Engelen 1995);

FIG. 18 shows a schematic diagram of plasmid pBNP58040 as used in thepresent invention. The plasmid contains the SmaI/SacI DNA fragment frompSK58040 inserted into the SmaI/SacI sites in pBINplus. This fragmentcontains the ohp operator from nucleotide 1036 to nucleotide 1449 of SEQID NO: 1 upstream of the CaMV 35S −90 bp core promoter, GUS intron(Vancanneyt et al., 1990) and nos terminator;

FIG. 19 shows a schematic diagram of plasmid pBNP60040 as used in thepresent invention. The plasmid contains the XhoI/SacI fragment frompSK60040 inserted into the XhoI/SacI sites in pBINplus. This fragmentcontains the chimeric promoter (Seq. ID. No: 13), a GUS intron(Vancanneyt et al., 1990) and a nos terminator,

FIG. 20 shows a schematic diagram of the plasmid pBNP10489 as used inthe present invention. The plasmid contains the HindIII/SacI fragmentfrom pSK10489 inserted into the HindIII/SacI sites in pBINplus. Thisfragment contains the ohpR sequence inserted between the CaMV 35Spromoter and the CaMV 35S terminator;

FIG. 21 shows a schematic diagram of the plasmid pBNP10490 as used inthe present invention. The plasmid contains the HindIII/SacI fragmentfrom pSK10490 inserted into the HindIII/SacI sites in pBINplus. Thisfragment contains the translational fusion between the ohpR codingsequence (nucleotide 295 to nucleotide 1035 of SEQ ID NO: 1) and part ofthe C1 cDNA (from the NarI at 536 bp to the end of the coding region atnucleotide 839, amino acids 179 to 279 of the C1 protein) insertedbetween the CaMV 35S promoter and the CaMV 35S terminator in pDV35S;

FIG. 22 shows a schematic diagram of the plasmid pBNP10491 as used inthe present invention. The plasmid contains the HindIII/SacI fragmentfrom pSK10491 inserted into the HindIII/SacI sites in pBINplus, Thisfragment contains the translational fusion between the ohpR codingsequence (nucleotide 295 to nucleotide 1035 of SEQ ID NO: 1) and part ofthe C1 cDNA (from the PstI site at nucleotide 674 to the end of thecoding region at nucleotide 839, amino acids 219 to 279 of the C1protein) inserted between the CaMV 35S promoter and the CaMV 35Sterminator in pDV35S;

FIG. 23 shows a schematic diagram of the plasmid pOH001 as used in thepresent invention. The plasmid is a double construct in pBINpluscontaining the ohp operator from nucleotide 1036 to nucleotide 1449 ofSEQ ID NO: 1 upstream of the CaMV 35S −90 bp core promoter, GUS intronand nos terminator, and also containing the ohpR sequence between theCaMV 35S promoter and the CaMV 35S terminator;

FIG. 24 shows a schematic diagram of the plasmid pOH003 as used in thepresent invention. The plasmid is a double construct in pBINpluscontaining the ohp operator from nucleotide 1036 to nucleotide 1449 ofSEQ ID NO: 1 inserted upstream of the CaMV 35S −90 bp core promoter, GUSintron and nos terminator, and also containing the translational fusionbetween the ohpR coding sequence (nucleotide 295 to nucleotide 1035 ofSEQ ID NO: 1) and part of the C1 cDNA (from the NarI at nucleotide 536to the end of the coding region at nucleotide 839, amino acids 179 to279 of the C1 protein) between the CaMV 35S promoter and the CaMV 35Sterminator,

FIG. 25 shows a schematic diagram of the plasmid pOH004 as used in thepresent invention. The plasmid is a double construct in pBINplus,containing the ohp operator from nucleotide 1036 to nucleotide 1449 ofSEQ ID NO: 1 upstream of the CaMV 35S −90 bp core promoter, GUS intronand nos terminator, and also containing the translational fusion betweenthe ohpR coding sequence (nucleotide 295 to nucleotide 1035 of SEQ IDNO: 1) and part of the C1 cDNA (from the PstI site at nucleotide 674 tothe end of the coding region at nucleotide 839, amino acids 219 to 279of the C1 protein) between the CaMV 35S promoter and the CaMV 35Sterminator;

FIG. 26 shows a schematic diagram of the plasmid pOH005 as used in thepresent invention. The plasmid is a double construct in pBINplus,containing the chimeric promoter (Seq. ID. 13), a GUS intron (Vancanneytet al., 1990) and a nos terminator, and also containing the ohpRsequence between the CaMV 35S promoter and the nos terminator and alsocontaining the ohpR sequence between the CaMV 35S promoter and the CaMV35S terminator;

FIG. 27 shows a schematic diagram of the plasmid pOH006 as used in thepresent invention. The plasmid is a double construct in pBINplus,containing the chimaeric promoter (Seq. ID. 13), a GUS intron(Vancanneyt et al., 1990) and a nos terminator, and also containing thetranslational fusion between the ohpR coding sequence (nucleotide 295 tonucleotide 1035 of SEQ ID NO: 1) and part of the C1 cDNA (from the NarIat nucleotide 536 to the end of the coding region at nucleotide 839,amino acids 179 to 279 of the C1 protein) between the CaMV 35S promoterand the CaMV 35S terminator;

FIG. 28 shows a schematic diagram of the plasmid pOH007 as used in thepresent invention. The plasmid is a double construct in pBINpluscontaining the chimeric promoter (Seq. ID. 1), a GUS intron (Vancanneytet al., 1990) and a nos terminator, and also containing thetranslational fusion between the ohpR coding sequence (nucleotide 295 tonucleotide 1035 of SEQ ID NO: 1) and part of the C1 cDNA (from the PstIsite at nucleotide 674 to the end of the coding region at nucleotide839, amino acids 219 to 279 of the C1 protein) between the CaMV 35Spromoter and the CaMV 35S terminator.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of controlling eukaryotic geneexpression comprising introducing into or transforming a eukaryotic cellwith (i) an inducible gene expression system, comprising a firstnucleotide sequence comprising a first 5′ regulatory region operablylinked to a nucleic acid sequence which encodes a regulator polypeptideand an untranslated 3′ termination sequence, and (ii) a secondnucleotide sequence comprising a second 5′ regulatory region operablylinked to a nucleic acid sequence which is a coding or noncodingsequence (i.e., the target gene or sequence), the expression of thenucleic acid sequence of the second nucleotide sequence being controlledby the regulator polypeptide of the first nucleotide sequence using aninducer. The inducer thereby causes modulation of expression of thenucleic acid sequence of the second nucleotide sequence (the targetgene). The nucleotide sequence of the regulator polypeptide and/or thesecond 5′ regulatory region, or parts thereof, of the second nucleotidesequence are preferably isolated from a prokaryote source.

While the first nucleotide sequence of the method and chimeric genehereof advantageously comprise an untranslated 3′ termination sequence,a termination sequence may not be essential to the operation of theinducible expression system.

Advantageously the inducible gene expression system is a chemicallyinducible gene expression system.

Preferably, one or more of the 5′ regulatory regions each comprises apromoter which allows expression in eukaryote cells and/or tissues.

Appropriate promoters are chosen so that expression of the regulatorpolypeptide may be constitutive, developmentally regulated,tissue-specific, cell-specific or cell compartment-specific. Suitableconstitutive promoters include but are not limited to CaMV 35S and CaMV19S promoters.

Suitable tissue specific promoters include but are not limited to thepatatin promoter and the petE promoter.

Suitable cell compartment promoters include but are not limited topromoters of chloroplast genes, such as the gene encoding the largesubunit of ribulose biphosphate carboxylase and promoters ofmitochondrial genes, such as the 18S-5S rRNA genes. Other suitablepromoters will be known to one skilled in the art.

Advantageously, the 5′ regulatory regions may also comprise one or moreenhancer sequences. The enhancer sequence may be a transcriptionaland/or translational enhancer sequence.

Numerous sequences have been found to enhance gene expression intransgenic plants. Suitable translational enhancer sequences include anumber of non-translated leader sequences derived from viruses are knownto enhance expression. Specifically, leader sequences from TobaccoMosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV) and AlfalfaMosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g. Gallie et al., 1987, Skuzeski et al., 1990). Otherleader sequences known in the art include but are not limited to:Picornavirus leaders, Potyvirus leaders, AMV RNA4 leader (Jobling &Gehrke 1987) or the HSP 70 leader (disclosed in U.S. Pat. No.5,659,122).

Suitable transcriptional enhancer sequences will be known to thoseskilled in the art, such as the petE enhancer disclosed in ourInternational Patent Application, Publication No. WO 97/20056.

Various intron sequences have been shown to enhance expression whenadded to the 5′ regulatory region. For example, the introns of the maizeAdh1 gene have been found to significantly enhance the expression of thewild-type gene under its cognate promoter when introduced into maizecells (Callis et al., 1987). International Patent Application,Publication No. WO 9319189 discloses the use of the Hsp70 intron frommaize to enhance gene expression in transgenic plants.

Advantageously, the regulator polypeptide comprises one or more domains,which domains may be a ligand binding domain, a nucleic acid bindingdomain, a transactivation domain, a targeting domain, asilencing/repressing domain or a dimerization domain. The regulatorsequence may thus comprise a chimeric gene of different sequences.

The ligand binding domain suitably comprises a sequence of amino acidswhose structure binds non-covalently a complementary ligand. The ligandmay be a chemical ligand. Hence, a ligand binding domain and its ligandform a complementary binding pair. Ligand binding domains for theconstruction of chimaeric regulator polypeptides may also be obtainedfrom a variety of sources. The complementary ligand may be the inducer,a derivative or a precursor of the inducer.

It is possible to use two or more chemical ligands that may act togetheras synergists and/or antagonists. The source of chemical ligand willdepend on which ligand binding domains are present in the regulatorpolypeptide. Any chemical compound will suffice as long as it is shownto form a complementary binding pair with the chosen ligand bindingdomain.

The nucleic acid binding domain comprises a sequence of amino acidswhich binds non-covalently to a specific nucleotide sequence known as aresponse element (RE).

The response element may be located in the 5′ regulatory region of thesecond nucleotide sequence. The nucleotide sequence and linearorientation determines which nucleic acid binding domain or domains willform a complementary binding pair with the response element.Considerable flexibility can be introduced into the method ofcontrolling gene expression by using these conserved response elementsin other ways.

Additional flexibility in controlling gene expression may be obtained byusing nucleic acid binding domains and response elements from othernucleic acid binding proteins, which include but are not limited to theLexA, Gal4, LacI, Tet, C1 and Ace1 proteins described above.

A further degree of flexibility in controlling gene expression can beobtained by combining response elements which form complementary bindingpairs with nucleic acid binding domains from different types of nucleicacid binding proteins, i.e. overlapping response elements.

The transactivation domain comprises one or more sequences of aminoacids acting as subdomains which affect the operation of transcriptionfactors during pre-initiation and assembly at the TATA box. The effectof the transactivation domain is to allow repeated transcriptioninitiation events, leading to greater levels of gene expression.Different transactivation domains are known to have different degrees ofeffectiveness in their ability to increase transcription initiation. Inthe present invention, it is desirable to use transactivation domainswhich have superior transactivating effectiveness in eukaryotic cells inorder to create a high level of target gene expression in eukaryoticcells. Transactivation domains which have been shown to be particularlyeffective include but are not limited to Vp16 (isolated from the herpessimplex virus) and C1 isolated from maize. Other transactivation domainsknown to those skilled in the art will also be effective.

The silencing/repressing domain comprises one or more sequences of aminoacids acting as subdomains which affect the RNA polymerase II basal orregulatory transcription machinery. The effect of thesilencing/repressing domain is to stop the progression of transcription.Different silencing/repressing domains are known to have differentdegrees of effectiveness in their ability to decrease transcription. Inthe present invention, it is desirable to use silencing/repressingdomains which have superior silencing/repressing effectiveness ineukaryotic cells in order to create a high level of target generepression in eukaryotic cells. Silencing/repression domains which havebeen shown to be particularly effective include but are not limited tothe KRAB domains identified in human, mouse and Xenopus zinc fingerproteins (for review see Hanna-Rose & Hansen 1996) and the Oshox1protein of rice (Meijer et al., 1997). Other silencing/repressingdomains known to those skilled in the art will also be effective.

The dimerization domain comprises one or more sequences of amino acidsacting as subdomains which affect the protein-protein interaction.Different dimerization domains are known to have different degrees ofeffectiveness in their ability to form protein-protein interactions. Inthe present invention, it is desirable to use dimerization domains whichhave superior dimerizafion effectiveness in eukaryotic cells in order tocreate a high level of protein-protein interaction in eukaryotic cells.Dimerization domains which have been shown to be particularly effectiveinclude but are not limited to Helix-loop-helix domains of Myc and MycoDand the leucine zipper domains of Myc and GCN4 proteins. Otherdimerization domains known to those skilled in the art will also beeffective.

The targeting domain may comprise targeting polypeptides to direct theregulator sequence to different parts of eukaryotic cells. Suitabletargeting domains include but are not limited to examples such as aplasma membrane targeting sequence (Hedley et al., 1993), golgi,endoplasmatic reticulum (Iturriaga et al., 1989), nuclear targetingsignals (Varagona et al., 1992, Raikhel 1992), chloroplast (Rensink etal., 1998), mitochondrial (Boutry et al., 1987) or inner envelopetargeting sequences (Knight & Gray 1995).

The nucleotide sequences which encode any of the above domains mayadvantageously be modified for improved expression in eukaryotes, havealtered functionality, or both. Such modifications include, but are notlimited to, altering codon usage, insertion of introns or creation ofmutations, preferably in the ligand binding domain and/or the nucleotidebinding domain. Modified nucleotide sequences of the regulatory sequenceare an aspect of the present invention.

Furthermore, ligand-binding, nucleic acid binding, transactivation andtargeting domains may be assembled in a chimeric regulator polypeptidein any functional arrangement.

Chimeric regulator polypeptides may also have multiple domains of thesame type, for example, more than one transactivation domain or nucleicacid binding domain per regulator polypeptide. Mutant regulatorpolypeptides may be prepared by methods of mutagenesis known in the art,such as chemical mutagenesis or site-directed mutagenesis. This mightresult in ligand binding domains with altered ligand binding and/ornucleic acid binding domains with altered recognition sites.

Advantageously the regulatory sequence comprises a ligand binding domainand/or a DNA binding domain. Preferably, the regulator sequence is thenucleotide sequence from 295-1035 bp of SEQ ID NO: 1. Advantageously thesequence may be isolated from the ohpR sequence in Rhodococcus sp. V49.Subsequences of this sequence having the necessary function may also beused in the invention.

Rhodococcus sp. V49 encodes the OHP catabolic operon, which is presentedin SEQ ID NO: 1, which sequence shows the nucleotide sequences amongothers of the ohpR, the ohpA operator region (1036-1260 bp), ohpA, OhpB,OhpC and OhpD genes, which when expressed allow growth on OHP as solecarbon-energy source. SEQ. ID. Nos. 2 through 7 represent amino acidsequences of the proteins encoded by the OHP catabolic operon, forexample, ohpR regulator (SEQ ID NO: 2), ohpA transport (SEQ ID NO: 3),OhpB monoxygenase (SEQ ID NO: 4), OhpD catechol 2,3-dioxygenase (SEQ IDNO: 5), and OhpC hydrolase (SEQ ID NO: 6). Nucleic acid sequencessubstantially similar to those sequences or nucleic acid sequencesencoding proteins with similar functionality may also be suitable foraspects of the present invention.

Gene sequence similarity is established by Southern Blot screening. Suchscreening is initially carried out under low-stringency conditions,which comprise a temperature of about 37° C. or less, a formamideconcentration of less than about 50%, and a moderate to low salt (e.g.Standard Saline Citrate (‘SSC’)=0.15 M sodium chloride; 0.15 M sodiumcitrate; pH7) concentration. Alternatively, a temperature of about 50°C. or less and a high salt (e.g. SSPE=0.280 mM sodium chloride; 9 mMdisodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mMsodium EDTA; pH 7.4). Preferably the screening is carried out at about37° C., a formamide concentration of about 20%, and a salt concentrationof about 5×SSPE. These conditions will allow the identification ofsequences which have a substantial degree of similarity with the probesequence, without requiring the perfect homology for the identificationof a stable hybrid. The phrase ‘substantial similarity’ refers tosequences which share at least 50% overall sequence identity.Preferably, hybridisation conditions will be selected which allow theidentification of sequences having at least 70% sequence identity withthe probe, while discriminating against sequences which have a lowerlevel of sequence identity with respect to the probe. After lowstringency hybridisation has been used to identify several bacterialwhose genome or DNA sub-clones exhibit a substantial degree ofsimilarity with the probe sequence, this subset of genomes or sub-clonesis then subjected to higher stringency hybridisation, so as to identifythose of this subset of genomes or sub-clones having a particularly highlevel of homology with respect to the probe sequences. Medium stringencyconditions comprise a temperature of about 39° C. and a medium salt(SSC) concentration. High stringency conditions comprise a temperatureof about 42° C. or less, and a low salt (SSC) concentration.Alternatively, they may comprise a temperature of 65° C. or less, and alow salt (SSPE) concentration. Preferred conditions for such screeningcomprise a temperature of about 42° C., a formamide concentration ofabout 20%, and a salt concentration of about 2×SSC, or a temperature ofabout 65° C., and a salt concentration of about 0.2 SSPE.

Suitable untranslated 3′termination sequences such as the CaMV 35S ornos terminator will be known to those skilled in the art.

Preferably, the 5′ regulatory region of the second nucleotide sequencemay also comprise a core promoter sequence and the response element (RE)or response elements necessary for complementary binding of theregulator polypeptide. By core promoter it is intended that the basalpromoter elements are inactive or nearly so without activation. Such apromoter has low background activity in eukaryotes when there is notransactivator present, or when enhancer or response element bindingsites are absent. Core promoters that are particularly useful for targetgenes in plants are the A1 core promoter which is obtained from the A1gene of maize (Tuerck & Fromm, 1994) or the CaMV35s core promoter.

Alternatively, the 5′ regulatory region of the second nucleotidesequence may also comprise a full-length promoter sequence and theresponse element (RE) or response elements necessary for complementarybinding of the regulator polypeptide. Such a promoter has high activityin eukaryotes when there is no transactivator present. Full-lengthpromoters that are particularly useful for target genes in plants arethe CaMV 35S promoter, the CERV promoter and the petE promoter.

Preferably, the response element of the 5′ regulatory region of thesecond nucleotide sequence is derived from the nucleotide sequence seenfrom nucleotide 295 to nucleotide 2805 in SEQ ID NO: 1. Advantageouslythe sequence is isolated from the ohpA promoter region (nucleotides1036-1260 of SEQ ID NO: 1) in Rhodococcus sp. V49 (ATCC19070).Subsequences of this sequence having the necessary function and/ormultiples of this sequence or a sequences can be used in the presentinvention in normal or reverse orientation, upstream or downstream ofthe core promoter, and in any order thereof.

Substantially similar sequences to the ohpR-ohpA region in accordancewith the hybridization conditions described above are also within thescope of the present invention. Suitable coding sequences in the secondnucleotide sequence include, but are not limited to, sequences whichencode proteins involved in carbon metabolism; flowering; fertilityand/or sterility, for example, the use of barnase or diptheria toxinA-chain; cell wall metabolism; genes that respond to environmentalsignals, for example pathogen attack, such as nematode, arachnid oraphid attack; or bacterium, fungus, virus, or insect resistance; orgenes that confer resistance to antibiotics, herbicides or other toxiccompounds.

The coding sequence may be homologous or heterologous in origin withrespect to the eukaryote being transformed.

Sense, co-suppression or anti-sense technology may be used as requiredto achieve alteration of the eukaryote. Nucleotide sequences may beintroduced into the cell by any method known to one skilled in the art.Transformation techniques such as the use of Agrobacterium,microinjection, microprojectile-bombardment, electroporation and othersknown to the skilled man are among those methods for which thisinvention is appropriate.

The expression of the nucleic acid sequence of the second nucleotidesequence (also known herein as the target gene) may be suitablyincreased or decreased, whether from a basal or medial levelrespectively, or completely repressed or activated.

Advantageously, an increase in target gene expression levels may becaused by the addition or presence of the inducer. Alternatively, anincrease in target gene expression levels may be caused by thewithdrawal or absence of the inducer. Similarly, a decrease in targetgene expression levels may be caused by the addition or presence of theinducer, or alternatively, a decrease in target gene expression levelsmay be caused by the withdrawal or absence of the inducer.

Preferably, the inducer which causes modulation of expression of thenucleic acid sequence is a chemical compound, such as OHP, 2-hydroxycinnamic acid, toluene, bezene, n-hexadecane or a functional equivalentof either. The inducer may also, however, be a protein or nucleic acidsequence, depending on the complementary domain of the regulatorsequence. The 5′ regulatory region of the second nucleotide sequence maysuitably comprise one or more response elements, each being necessaryfor complementary binding of an appropriate domain or other portion ofthe regulator sequence.

Advantageously the inducer acts by indirect action. Alternatively, theinducer acts by direct action.

Preferably the eukaryotic cell is a plant cell. The plant cell may beone or more from the group consisting of, for example, crops such aspotato, wheat, maize, barley, tomato, rice, canola, sugarbeet ortobacco; trees such as eucalyptus species, populus or malus; or otherplants, such as Arabidopsis.

Preferably the gene expression system comprises a single constructcontaining the first nucleotide sequence and the second nucleotidesequence. In the alternative, the gene expression system may utilise twoor more separate constructs, and further each construct may beintroduced into separate eukaryotes, which are then transferred into oneeukaryote, biologically mated or crossed, for example, to bring theconstructs together.

Alternatively, the expression system may comprise one transformationstep followed by a further transformation step or steps. Each step mayintroduce one or more additional constructs, for example,co-transformation or re-transformation.

The present invention also provides a chimeric gene comprising a firstnucleotide sequence comprising a first 5′ regulatory region operablylinked to a nucleic acid sequence which encodes a regulator polypeptideand an untranslated 3′ termination sequence, and a second nucleotidesequence comprising a second 5′ regulatory region operably linked to anucleic acid sequence which is a coding or non-coding sequence (i.e.,target gene or sequence), the expression of the nucleic acid sequence ofthe second nucleotide sequence being controlled by the regulatorpolypeptide of the first nucleotide sequence using an inducer. Theinducer thereby causes modulation of expression of the nucleic acidsequence of the second nucleotide sequence (the target gene). Thenucleotide sequence of the regulator polypeptide and/or the second 5′regulatory region or parts thereof of the second nucleotide sequence areisolated preferably from a prokaryote source.

Alternatively there may be provided a first chimaeric gene comprisingthe first nucleotide sequence and a second chimaeric gene comprising thesecond nucleotide sequence.

Advantageously the chimaeric gene is utilised in a plasmid, vector orother transportable medium suitable for microbiological genetictransformation.

Plant tissue, such as cells, organs, seed and other plant partstransformed using the aspects of the present invention are also aspectsof the instant invention.

6. EXAMPLES

In order to transform eukaryotes the preparation of constructs and theuse of transformation techniques are required in accordance with thefollowing Examples.

6.1 Materials and Methods

Generally speaking, those skilled in the art are well able to constructvectors and design protocols for recombinant gene expression in commonhosts such as E. coli and Agrobacterium. Suitable vectors for theconstruction of gene expression cassettes can be chosen or constructed,containing appropriate regulatory sequences, including promotersequences, terminator fragments, polyadenylation sequences, enhancersequences, marker genes and other sequences as appropriate. For furtherdetails, see, for example, Molecular Cloning: A Laboratory manual: 2ndedition, Sambrook et al. 1989, Cold Spring Harbor Laboratory Press. Manyknown techniques and protocols for manipulation of nucleic acid, forexample in preparation of nucleic acid constructs, mutagenesis,sequencing and, introduction of DNA into cells, gene expression, andanalysis of proteins are described in detail in Current Protocols inMolecular Biology, Second Edition, Ausubel et al., eds, John Wiley andSons 1992. The disclosures of Sambrook et al., and Ausubel et al., areincorporated herein by reference.

However the present inventors have recognized that certain methodspreviously employed in the art which were developed for enteric bacteriasuch as E. coli may not be most appropriate for use in plant geneticconstructs. Accordingly, advantageous methods have been developed by theinventors which in preferred forms allow the rapid construction of OHPgenetic constructs and operably linked inducible 5′ regulatory regionsand regulator constructs.

The following examples further describe the materials and methods usedin carrying out the invention and the subsequent results. They areoffered by way of illustration, and their recitation should not beconsidered as a limitation of the claimed invention.

6.2 Isolation of the ohpR sequence

Example 1

The coding sequence of the OHP operon OhpR (from nucleotide 295 tonucleotide 1035) was amplified by PCR from construct pJP58 using theprimers OHPR3 (SEQ ID NO: 8) and OHPR4 (SEQ ID NO: 9). The constructpJP58 was deposited by Advanced Technologies (Cambridge) Ltd of 210Cambridge Science Park, Cambridge CB4-0WA, England under the BudapestTreaty on the International Recognition of the Deposit ofMicro-organisms for the purposes of Patent Procedure at the NationalCollection of Industrial and Marine Bacteria (NCIMB), 23 St. MacharStreet, Aberdeen, Scotland on 21^(st) Dec. 1998 under accession numberNCIMB 40997. It contains a 2 kb BamHI fragment encoding the ohpA-ohpRregion (nucleotides 1-1869 of SEQ ID NO: 1) cloned into pUC19 using theunique Bam site (Veira J. & Messing, J. 1982).

The PCR product was restriction digested with EcoRI and NotI and clonedinto pBluescript (Stratagene) also digested with EcoRI and NotI. Theresulting plasmid was named pSK489 (FIG. 1) and sequenced.

6.3 Isolation of the transcriptional activator sequence C1

Example 2

The C1 cDNA region was amplified by PCR from plasmid p35SC1 (FIG. 2), asdescribed in Tuerck & Fromm (1994), using the primers C11 (SEQ ID NO:10) and C12 (SEQ ID NO: 11). The PCR product was digested with EcoRI andNotI and ligated into pBluescript digested with EcoRI and NotI. Theresulting plasmid was named pSK483 (FIG. 3) and sequenced.

6.4 Isolation of the Operator Sequence

Example 3

Part of the operator region of the OHP operon (from nucleotide 1036 tonucleotide 1449 in SEQ ID NO: 1) was amplified by PCR from constructpJP58 using the oligonucleotide primers op1 (SEQ ID NO: 12) and op2 (SEQID NO: 13). The 441 bp PCR product was restriction digested to with XhoIand SalI, gel-purified and ligated into pBluescript digested with XhoIand SalI. The resulting plasmid was named pSK-59 (FIG. 4) and sequenced.

6.5 Construction of the Construct for the Nucleic Acid Sequence in theSecond Nucleotide Sequence

Example 4

The plasmid pSK-59 (FIG. 4) was digested with XhoI and SalI, the 414 bpoperator region was gel-purified and ligated with pBS52040 (FIG. 5)which had been digested with XhoI and phosphatased. The resultingplasmid was named pSK58040 (FIG. 6).

6.6 Construction of a Chimeric CaMV35S Promoter-ohp Regulator Construct

Example 5

The three oligonucleotides CaMVop2 (SEQ ID NO: 14), CaMVop3 (SEQ ID NO:15) and CaMVop4 (SEQ ID NO:16) were annealed in equimolar amounts (500pmole each primer) and diluted tenfold. 5 μl of this dilution were usedas a template for a PCR reaction (50 μl total) catalysed by aproof-reading Taq polymerase to generate double stranded product. ThePCR product was resolved on an 8% polyacrylamide gel. The 125 bp PCRproduct was excised and purified using techniques described in Sambrooket al (1989). 1 μl of the total eluted double stranded DNA solution (50μl) was used as a template in a PCR reaction (50 μl total) primed byoligonucleotide primers CaMVopF1 (SEQ ID NO: 17) and CaMVopR1 (SEQ IDNO: 18) and catalysed by a proof-reading Taq polymerase. The PCR productfrom this reaction was digested to completion with EcoRV and BamHI andthe 133 bp restriction fragment ligated with plasmid pDV35S1 (FIG. 7)similarly digested to completion with EcoRV and the resulting constructwas named pDV60 (FIG. 8). The inserted region was sequenced. PlasmidpDV60(FIG. 8) was digested with XhoI and BamHI. The 476 bp syntheticpromoter restriction fragment (SEQ ID NO: 19) was gel purified asdescribed above and ligated into pSK52040 (FIG. 5) similarly digestedwith XhoI and BamHI. This plasmid was named pSK60040 (FIG. 9). Thechimeric promoter in SEQ ID NO: 18 contains a 36 bp region of the ohpoperon (from nucleotide 1225 to nucleotide 1260) inserted into the CaMV35S promoter at nucleotide 21.

6.7 Construction of Chimeric Regulator Sequences

Example 6

The plasmid pSK483 (FIG. 3) was digested to completion with PstI andXbaI. The 162 bp fragment (the C1 cDNA region from amino acids 219 to273 of the C1 protein) was gel-purified and ligated with pSK489 (FIG. 1)similarly digested with PstI and XbaI. The resulting plasmid was namedpSK491 (FIG. 11). This ligation results in a translation fusion of theOHPR nucleotide sequence and the C1 nucleotide sequence for thetranscriptional activation domain from amino acid 219 to 273.

The plasmid pSK483 (FIG. 3) was also digested to completion with NarIand XbaI. The 303 bp fragment of the C1 cDNA region (encoding aminoacids 173 to 273 of the C1 protein) was gel-purified and ligated withpSK489 (FIG. 1) similarly digested with NarI and XbaI. The resultingplasmid was named pSK490 (FIG. 10). This ligation results in atranslation fusion of the ohpR nucleotide sequence and the C1 nucleotidesequence for the transcriptional activation domain from amino acid 173to 273.

6.8 Construction of pDV35S2

Example 7

pDV35S1 (FIG. 7) was digested with HindIII and SacI and the 668 bpfragment containing the CaMV 35S promoter/terminator was gel-purifiedand ligated with pUCAP (FIG. 12) which was digested with HindIII andSacI. The resulting construct was named pDV35S2 (FIG. 13).

6.9 Construction of a Regulator Expression Construct

Example 8

Plasmids pSK489 (FIG. 1), pSK490 (FIG. 10) and pSK491 (FIG. 11) weredigested with BamHI and XbaI, the fragments encoding the regulatorsequences were gel-purified and ligated with pDV35S1 (FIG. 7), similarlydigested with BamHI and XbaI. The resulting plasmids were named pSK10489(ohpR, FIG. 14), pSK10490 (ohpR-C1 NarI/XbaI fusion, FIG. 15) andpSK10491 (ohpR-C1 PstI/XbaI fusion, FIG. 16) respectively.

6.10 Construction of pBNP58040

Example 9

Plasmid pSK58040 (FIG. 6) was digested to completion with HindIII andSmaI and the 2837 bp fragment containing the CaMV 35S promoter-GUS-nosterminator was gel-purified and ligated into pBINplus (FIG. 17)similarly digested with HindIII and SmaI. The resulting plasmid wasnamed pBNP58040 (FIG. 18).

6.11 Construction of pBNP60040

Example 10

Plasmid pSK60040 (FIG. 9) was digested to completion with HindIII andSacI and the promoter-Gus fragment was gel-purified and ligated withpBINplus (FIG. 17) similarly digested with HindIII and SacI. Theresulting plasmid was named pBNP60040 (FIG. 19).

6.12 Construction of Plant Transformation Vectors Carrying the RegulatorGenes.

Example 11

The regulator cassettes were cut out of pSK10489 (FIG. 14), pSK10490(FIG. 15), and pSK10491 (FIG. 16), respectively. DNA was digested withHindIII and SacI. The restriction fragments containing the CaMV 35Spromoter-regulator were gel-purified. The isolated fragments wereligated with pBINplus (FIG. 17) similarly digested with HindIII andSacI. The resulting plasmids were named pBNP10489 (FIG. 20—pBNPcontaining 10489, ohpR), pBNP10490 (FIG. 21—pBNP containing 10490,ohpR-C1 NarI fusion) and pBNP10491 (FIG. 22—pBNP containing 10491,ohpR-C1 PstI fusion).

6.13 Construction of Double Gene Expression Constructs

Example 12

The CaMV 35S promoter-regulator fragments were cut out of pSK10489 (FIG.14), pSK10490 (FIG. 15) and pSK10491 (FIG. 16) respectively. DNA wasdigested with NotI, blunt-ended with Klenow DNA polymerase and thendigested with HindIII. The restriction fragments containing the CaMV 35Spromoter/regulator were gel-purified. pBNP58040 FIG. 18) and pBNP60040(FIG. 19) were digested with HindIII and SmaI. The gel-purifiedfragments were ligated with either the digested pBNP58040 (FIG. 18) orthe digested pBNP60040 (FIG. 19). The resulting plasmids were namedpOH001 (FIG. 23—pBNP containing 58040 and 10489), pOH003 (FIG. 24—pBNPcontaining 58040 and 10490), pOH004 (FIG. 25—pBNP containing 58040 and10491), pOH005 (FIG. 26—pBNP containing 60040 and 10489), pOH006 (FIG.27—pBNP containing 60040 and 10490) and pOH007 (FIG. 28—pBNP containing60040 and 10491).

6.14 Transformation of Agrgobacterium

Example 13

The plant transformation vectors (as described in Examples 9-12, FIGS.18-28) were electroporated into Agrobacterium tumefaciens cells.Agrobacterium cultures were selected on kanamycin-containing medium (50μg/ml). The cultures were grown in liquid medium and then used for thetransformation of plant species.

6.15 Transformation or Retransformation of Plants

Example 14

Tobacco and potato plants can be transformed using the method of leafdisk cocultivation as essentially described by Horsch et al., (1985).The binary vectors as described above in Examples 9-12 (FIGS. 18-28) aretransferred to Agrobacterium tumefaciens LBA4404 using the method ofelectroporation, and cultures of said Agrobacteria can be used intransformation so that regenerated plants carry the chimeric genes asdescribed in Examples 9-12.

Young leaves were dissected under sterile conditions, from approximately4 week old Eucalyptus species cultures grown in Magenta boxes (7 cm×7cm×13 cm) on LS media at 25 C, in a growth room in our tissue culturelaboratory and used for Agrobacterium-mediated transformation (Horsch etal. 1985) using the strain EHA105.

Inoculated tissue was left to co-cultivate for 4 days on LS media (plus20-g/l glucose, 0.7% agarose, 0.1 mM Zeatin and 1 μM NAA) in diffuselight in a growth, conditions as before.

Transformants were selected on 50 mg/ml kanamycin and 250 mg/mlclaforan. Arabidopsis thaliana was transformed following the protocolsfrom Bechthold et al., (1993) and Clough (1998). Plants were grown in agrowth cabinet at 22° C. under 18 h daylight before and aftervacuum-infiltration.

Several direct gene transfer procedures have been developed to transformplants and plant tissues without the use of an Agrobacteriumintermediate. In the direct transformation of protoplasts the uptake ofexogenous genetic material into a protoplast may be enhanced by use of achemical agent or electric field. The exogenous material may then beintegrated into the nuclear genome (Pazkowski et al., 1984, Potrykus etal., 1985). Alternatively, exogenous DNA can be introduced into cells orprotoplasts by microinjection. A solution of plasmid DNA is injecteddirectly into the cell with a finely pulled glass needle (Reich et al.,1986). A more recently developed procedure for direct gene transferinvolves bombardment of cells by microprojectiles carrying DNA (Klein etal., 1987). In this procedure tungsten or gold particles coated with theexogenous DNA are accelerated towards the target cells, resulting intransient expression and also in stable integration of the DNA into theplant genome.

Following transformation, the transformed cell or plant tissue isselected or screened by conventional techniques. The transformed cell orplant tissue contains the chimeric DNA sequences discussed above and isthe regenerated, if desired, by known procedures. The regenerated plantsare screened for transformation by standard methods. Progeny of theregenerated plants is continuously screened and selected for thecontinued presence of the integrated DNA sequence in order to developimproved plant and seed lines. The DNA sequence can be moved into othergenetic lines by a variety of techniques, including classical breeding,protoplast fusion, nuclear transfer and chromosome transfer.

The chimeric binary vector plasmids mentioned above can be used totransform a plant already carrying other chimeric genes by the methodsdescribed above.

6.16 Transient Expression

Example 15

Transient expression assays of the gene expression cassette wasessentially performed as described by Kapila et al., (1997), Rossi etal., (1993), Twell et al., (1989), Goff et al., (1990), Roth et al.,(1991) and Tuerck et al., (1994).

Leaf discs of 4-6 weeks old plants were excised and incubated with theAgrobacterium suspension. The discs were incubated for 1-5 days on wetWhatman paper before they were stained for GUS expression before andafter induction.

6.17 Induction of Reporter Gene Activity in Transgenic Plants

Example 16

OHP was applied to the plants (or plant cells) as a paint, spray or inthe medium in concentrations ranging from 0.01 mM to 100 mM in water orin 10 mM MES, pH5.6. Tissue was harvested prior to inducer applicationand at appropriate times after the application. The sample tissue wasground in extraction buffer and assayed for GUS reporter gene activityas described by Jefferson (1987). Tissue was also stained for GUSexpression as described by Jefferson (1987).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

REFERENCES

The following references are incorporated into the specification byreferences in their entireties.

-   Aoyama, T. & Chua, N.-H. (1997) Plant J. 11: 605-612.-   Boutry, M., Nagy, F., Poulsen, C., Aoyagi, K. & Chua, N.-H. (1987)    Nature 328:340-342.-   Bechthold, N., Ellis, J., Pelletier, G. (1993). Comptes Rendus de    l'Academie des Science Serie III Science de la Vie: 316: 1194-1199-   Caddick, M. X., Greenland, A. J., Jepson, I., Krause, K.-P., Qu, N.,    Riddell, K. V., Salter, M. G., Schuch, W., Sonnewald, U. &    Tomsett, A. B. (1998) Nature Biotechnol 16: 177-180.-   Clough, S. J. & Bent, A. F. (1998). Plant J. 16(6):735-743.-   Felenbok, B. (1991) J. Biotechnol. 17: 11-18.-   Fillinger, S. & Felenbok, B. (1996) Mol. Microbiol. 20: 475-488.-   Frohberg, C., Heins, L. & Gatz, C. (1991) PNAS 88: 10470-10474.-   Gallie et al. (1987) NAR 15: 8693-8711.-   Gatz, C. & Quail, P. H. (1988). PNAS 85: 1394-1397.-   Gatz, C., Kaiser, A. & Wendenburg, R. (1991) Mol. Gen. Genet. 227:    229-237.-   Gatz, C., Frohberg, C. & Wendenburg, R. (1992) Plant J. 2: 397-404.-   Goff, S. A., Klein, T. M., Roth, B. A., Fromm, M. E., Cone, K. C.,    Radicella, J. P. & Chandler, Y. L. et al. (1990). EMBO J.    9:2517-2522.-   Gossen, M., Freundlieb, S., Bender, G., Mueller, G., Hillen, W. &    Bujard, H. (1995) Science 268: 1766-1769.-   Hanna-Rose, W., & Hnasen, U. (1996). TIG 12(6):229-234.-   Haydon, D. J. & Guest, J. R. (1991). FEMS Microbiol. Letters    79:291-296.

Hedley, P. E., Machray, G. C. Davies, H. V., Burch, L. & Waugh, R.(1993) Plant Mol. Biol. 22:917-922.

-   Horsch, R. B., Fry, J. E., Hoffmann, N., Eichholtz, D.,    Rogers, S. G. & Fraley, R. T. (1985). Science 227:1229-1231.-   Iturriaga, G., Jefferson, R. A. & Bevan, M. W. (1989) Plant C.    1:381-390.-   Jefferson, R. A. (1987) Plant Mol. Biol. Rep. 5:387-405.-   Jobling, S. A. & Gehrke, L. (1987). Nature 325:622-625.-   Kapila, J., De Rycke, R., Van Montagu, M & Angenon, G. (1997). Plant    Science 122:101-108.-   Klein, T. M., Wolf, E. D., Wu, R. & Sanford, J. C. (1987). Nature    327:70-73.-   Knight, J. S. & Gray, J. C. (1995) Plant C. 7:1421-1432.-   Kulmburg, P., Judewicz, N., Mathieu, M., Lenouvel, F., Sequeval, D.    & Felenbrok, B. (1992) J. Biol. Chem. 267: 21146-21153.-   Lloyd, A. M., Schena, M., Walbot, V. & Davies, R. W. (1994) Science    266: 43-439.-   Lüscher, B. & Eisenman, R. N. (1990). Genes & Dev. 4:2025-2035.-   McKenzie, M. J., Mett, V. Reynolds, P. H. S. & Jameson, P. E. (1998)    Plant Physiol. 116: 969-977.-   Meijer, A. H., Scarpella, E., vam Dijk, E. L., Qin, L., Taal, A. J.    C., Rueb, S., McCouch, S. R., Schilperoort, R. A. & Hoge, J. H. C.    (1997). Plant J. 11(2):263-276.-   Mett, V. L., Lochhead, L. P. & Reynolds P. H. S. (1993) PNAS 90:    4567-4571.-   Paz-Ares, X., Ghosal, D., Wienaud, U., Peterson, P. A. & Saedler, H.    (1987). EMBO J. 6(12):3553-3558.-   Pazkowski, J., Shilito, RD., Saul, M. W., Mandak, V., Hohn. T,    Hohn, B. & Potrykus, I. (1984). EMBO J. 3:2717-2722.-   Picard, D., Salser, S. J. & Yamamoto, K. R. (1988). Cell 54:    1073-1080.-   Potrykus, I., Saul, M. W., Petruska, J., Pazkowski, J.,&    Shilito, R. D. (1985). Mol. Gen. Gen. 199:178-182.-   Raikhel, N. V. (1992) Plant Physiol. 100: 1627-1632.-   Reich, T. J. et al. (1986) Bio/Technology 4: 1001.-   Rensink, W. A., Pilon, M. & Weisbeek, P. (1998) Plant Physiol.    118:691-699.-   Roeder, F. T., Schmulling, T. & Gatz, C. (1994). Mol. Gen. Gen.    243:32-38.-   Rossi, L., Escudero, J., Hohn, B. & Tinland, B. (1993). Plant Mol.    Biol. Rep. 11: 220-229.-   Roth, B. A., Goff, S. A., Klein, T. M. & Fromm, M. E. (1991). The    Plant Cell 3:317-325.-   Salter, M. G., Paine, J. A., Riddell, K. V., Jepson, I.,    Greenland, A. J., Caddick, M. X. & Tomsett, A. B. (1998) Plant J.    16:127-132.-   Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning, A    laboratory Manual, Second Edition. Cold Spring Harbour Laboratory    Press 1989-   Schena, M., Lloyd, A. M. & Dacis, R. W. (1991) PNAS 88 10421-10425.-   Skuzeski, J. M., Nichols, L. M. & Gestelande, R. F. (1990). Plant    Mol. Biol. 15(1):65-79.-   Sommer, S., Siebert, M., Bechthold, A. & Heide, L. (1998) Plant Cell    Rep. 17: 891-896.-   Tuerck & Fromm (1994). Plant Cell 6:1655-1663.-   Twell, D., Klein, T. M., Fromm, M. E. & McCormick (1989) Plant    Physiol. 91: 1270-1274.-   van Engelen, F. A., Molthoff, J. W., Conner, A. J., Nap, J.-P.,    Pereira, A. & Steikema, W. J. (1995) Transgen. Res 4:288-290.-   Varagona, M. J., SchmidtR. J. & Raikhel, N. V. (1992) Plant C.    4:1213-1227.-   Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, Willmitzer, L &    Rocha-Sosa (1990). Mo. Gen. Genet. 220:245-250.-   Veira J. & Messing J. (1982) Gene 19: 259-268-   Weinmann, P., Gossen, M., Hillen, W., Bujard, H. & Gatz, C. (1994)    Plant J. 5(4): 559-569.-   Wilde, R. J., Shufflebottom, D., Cooke, S., Jasinska, I.,    Merryweather, A., Beri, R., Brammar, W. J., Bevan, M. &    Schuch, W. (1992) EMBO J. 11(4): 1251-1259.

1. A chimeric gene comprising a first nucleotide sequence comprising a5′ regulatory region operably linked to a nucleic acid sequence whichencodes a regulator polypeptide and an untranslated 3′ terminationsequence, and a second nucleotide sequence comprising a 5′ regulatoryregion operably linked to a nucleic acid sequence which is a coding ornon-coding sequence, the expression of said nucleic acid sequence ofsaid second nucleotide sequence being controlled by said regulatorpolypeptide of said first nucleotide sequence using an inducer, saidinducer thereby causing modulation of expression of said nucleic acidsequence of said second nucleotide sequence, and said nucleotidesequence of said regulator polypeptide and/or said 5′ regulatory regionor parts thereof of said second nucleotide sequence being isolated froma prokaryote source.
 2. A chimeric gene according to claim 1, whereinone or more of said 5′ regulatory regulatory regions each comprises apromoter which allows expression in eukaryote cells and/or tissues.
 3. Achimeric gene according to claim 1 or 2, wherein the promoter of said 5′regulatory region operably linked to said nucleic acid encoding saidregulator polypeptide is a constitutive, developmentally regulated,tissue-specific, cell-specific or cell compartment-specific promoter. 4.A chimeric gene according to claim 3, wherein said constitutive promoteris the CaMV 35S or CaMV 19S promoter.
 5. A chimeric gene according toclaim 3, wherein said tissue-specific promoter is the patatin promoteror the petE promoter.
 6. A chimeric gene according to claim 3, whereinsaid cell compartment promoter is a chloroplast gene promoter or amitochondrial gene promoter. 7-37. (Canceled)
 38. A method ofcontrolling eukaryotic gene expression comprising introducing into aeukaryotic cell with an inducible gene expression system, said induciblegene expression system comprising a first nucleotide sequence comprisinga 5′ regulatory region operably linked to a nucleic acid sequence whichencodes a regulator polypeptide and an untranslated 3′ terminationsequence, and a second nucleotide sequence comprising a 5′ regulatoryregion operably linked to a nucleic acid sequence which is a coding ornon-coding sequence, the expression of said nucleic acid sequence ofsaid second nucleotide sequence being controlled by the regulatorpolypeptide of the first nucleotide sequence using an inducer, saidinducer thereby causing modulation of expression of said nucleic acidsequence of said second nucleotide sequence, and said nucleotidesequence of said regulator polypeptide and/or said 5′ regulatory region,or parts thereof, of said second nucleotide sequence being isolated froma prokaryote source.
 39. A method according to claim 38, wherein saidinducible gene expression system is a chemically inducible geneexpression system.
 40. A method according to claim 38 or 39, whereinsaid coding sequence is homologous or heterologous in origin withrespect to the eukaryote being transformed.
 41. A method according toclaim 38 or 39, wherein expression of said nucleic acid sequence of saidsecond nucleotide sequence, said second nucleotide sequence being atarget gene, is increased or decreased, whether from a basal or mediallevel respectively, or completely repressed or activated.
 42. A methodaccording to claims 38 or 39, wherein an increase in target geneexpression levels is caused by the addition or presence of said inducer.43-58. (Canceled)
 59. Plant tissue transformed in accordance with themethod of claims
 38. 60-61. (Canceled)